MicroRNA molecules

ABSTRACT

In  Caenorhabditis elegans , lin-4 and let-7 encode 22- and 21-nucleotide RNAs, respectively, that function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as “small temporal RNAs” (stRNAs). We show that many more 21- and 22-nt expressed RNAs, termed microRNAs, (miRNAs), exist in invertebrates and vertebrates, and that some of these novel RNAs, similar to let-7 stRNA, are also highly conserved. This suggests that sequence-specific post-transcriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 35 USC § 371 National Phase Entry Application fromPCT/EP02/10881, filed Sep. 27, 2002, and designating the U.S.

The present invention relates to novel small expressed (micro)RNAmolecules associated with physiological regulatory mechanisms,particularly in developmental control.

In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotideRNAs, respectively (1, 2), that function as key regulators ofdevelopmental timing (3–5). Because the appearance of these short RNAsis regulated during development, they are also referred to as“microRNAs” (miRNAs) or small temporal RNAs (stRNAs) (6). lin-4 andlet-21 are the only known miRNAs to date.

Two distinct pathways exist in animals and plants in which 21- to23-nucleotide RNAs function as post-transcriptional regulators of geneexpression. Small interfering RNAs (siRNAs) act as mediators ofsequence-specific mRNA degradation in RNA interference (RNAi) (7–11)whereas miRNAs regulate developmental timing by mediatingsequence-specific repression of mRNA translation (3–5). siRNAs andmiRNAs are excised from double-stranded RNA (dsRNA) precursors by Dicer(12, 13, 29), a multidomain RNase III protein, thus producing RNAspecies of similar size. However, siRNAs are believed to bedouble-stranded (8, 11, 12), while miRNAs are single-stranded (6).

We show that many more short, particularly 21- and 22-nt expressed RNAs,termed microRNAs (miRNAs), exist in invertebrates and vertebrates, andthat some of these novel RNAs, similar to let-7 RNA (6), are also highlyconserved. This suggests that sequence-specific post-transcriptionalregulatory mechanisms mediated by small RNAs are more general thanpreviously appreciated.

The present invention relates to an isolated nucleic acid moleculecomprising:

-   -   (a) a nucleotide sequence as shown in Table 1, Table 2, Table 3        or Table 4    -   (b) a nucleotide sequence which is the complement of (a),    -   (c) a nucleotide sequence which has an identity of at least 80%,        preferably of at least 90% and more preferably of at least 99%,        to a sequence of (a) or (b) and/or    -   (d) a nucleotide sequence which hybridizes under stringent        conditions to a sequence of (a), (b) and/or (c).

In a preferred embodiment the invention relates to miRNA molecules andanalogs thereof, to miRNA precursor molecules and to DNA moleculesencoding miRNA or miRNA precursor molecules.

Preferably the identity of sequence (c) to a sequence of (a) or (b) isat least 90%, more preferably at least 95%. The determination ofidentity (percent) may be carried out as follows:I=n:Lwherein I is the identity in percent, n is the number of identicalnucleotides between a given sequence and a comparative sequence as shownin Table 1, Table 2, Table 3 or Table 4 and L is the length of thecomparative sequence. It should be noted that the nucleotides A, C, Gand U as depicted in Tables 1, 2, 3 and 4 may denote ribonucleotides,deoxyribonucleotides and/or other nucleotide analogs, e.g. syntheticnon-naturally occurring nucleotide analogs. Further nucleobases may besubstituted by corresponding nucleobases capable of forming analogousH-bonds to a complementary nucleic acid sequence, e.g. U may besubstituted by T.

Further, the invention encompasses nucleotide sequences which hybridizeunder stringent conditions with the nucleotide sequence as shown inTable 1, Table 2, Table 3 or Table 4, a complementary sequence thereofor a highly identical sequence. Stringent hybridization conditionscomprise washing for 1 h in 1×SSC and 0.1% SDS at 45° C., preferably at48° C. and more preferably at 50° C., particularly for 1 h in 0.2×SSCand 0.1% SDS.

The isolated nucleic acid molecules of the invention preferably have alength of from 18 to 100 nucleotides, and more preferably from 18 to 80nucleotides. It should be noted that mature miRNAs usually have a lengthof 19–24 nucleotides, particularly 21, 22 or 23 nucleotides. The miRNAs,however, may be also provided as a precursor which usually has a lengthof 50–90 nucleotides, particularly 60–80 nucleotides. It should be notedthat the precursor may be produced by processing of a primary transcriptwhich may have a length of >100 nucleotides.

The nucleic acid molecules may be present in single-stranded ordouble-stranded form. The miRNA as such is usually a single-strandedmolecule, while the mi-precursor is usually an at least partiallyself-complementary molecule capable of forming double-stranded portions,e.g. stem- and loop-structures. DNA molecules encoding the miRNA andmiRNA precursor molecules. The nucleic acids may be selected from RNA,DNA or nucleic acid analog molecules, such as sugar- orbackbone-modified ribonucleotides or deoxyribonucleotides. It should benoted, however, that other nucleic analogs, such as peptide nucleicacids (PNA) or locked nucleic acids (LNA), are also suitable.

In an embodiment of the invention the nucleic acid molecule is an RNA-or DNA molecule, which contains at least one modified nucleotide analog,i.e. a naturally occurring ribonucleotide or deoxyribonucleotide issubstituted by a non-naturally occurring nucleotide. The modifiednucleotide analog may be located for example at the 5′-end and/or the3′-end of the nucleic acid molecule.

Preferred nucleotide analogs are selected from sugar- orbackbone-modified ribonucleotides. It should be noted, however, thatalso nucleobase-modified ribonucleotides, i.e. ribonucleotides,containing a non-naturally occurring nucleobase instead of a naturallyoccurring nucleobase such as uridines or cytidines modified at the5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosinesand guanosines modified at the 8-position, e.g. 8-bromo guanosine; deazanucleotides, e.g. 7-deaza-adenosine; O- and N-alkylated nucleotides,e.g. N6-methyl adenosine are suitable. In preferred sugar-modifiedribonucleotides the 2′-OH-group is replaced by a group selected from H,OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN, wherein R is C₁–C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I. In preferredbackbone-modified ribonucleotides the phosphoester group connecting toadjacent ribonucleotides is replaced by a modified group, e.g. ofphosphothioate group. It should be noted that the above modificationsmay be combined.

The nucleic acid molecules of the invention may be obtained by chemicalsynthesis methods or by recombinant methods, e.g. by enzymatictranscription from synthetic DNA-templates or from DNA-plasmids isolatedfrom recombinant organisms. Typically phage RNA-polymerases are used fortranscription, such as T7, T3 or SP6 RNA-polymerases.

The invention also relates to a recombinant expression vector comprisinga recombinant nucleic acid operatively linked to an expression controlsequence, wherein expression, i.e. transcription and optionally furtherprocessing results in a miRNA-molecule or miRNA precursor molecule asdescribed above. The vector is preferably a DNA-vector, e.g. a viralvector or a plasmid, particularly an expression vector suitable fornucleic acid expression in eukaryotic, more particularly mammaliancells. The recombinant nucleic acid contained in said vector may be asequence which results in the transcription of the miRNA-molecule assuch, a precursor or a primary transcript thereof, which may be furtherprocessed to give the miRNA-molecule.

Further, the invention relates to diagnostic or therapeutic applicationsof the claimed nucleic acid molecules. For example, miRNAs may bedetected in biological samples, e.g. in tissue sections, in order todetermine and classify certain cell types or tissue types ormiRNA-associated pathogenic disorders which are characterized bydifferential expression of miRNA-molecules or miRNA-molecule patterns.Further, the developmental stage of cells may be classified bydetermining temporarily expressed miRNA-molecules.

Further, the claimed nucleic acid molecules are suitable for therapeuticapplications. For example, the nucleic acid molecules may be used asmodulators or targets of developmental processes or disorders associatedwith developmental dysfunctions, such as cancer. For example, miR-15 andmiR-16 probably function as tumor-suppressors and thus expression ordelivery of these RNAs or analogs or precursors thereof to tumor cellsmay provide therapeutic efficacy, particularly against leukemias, suchas B-cell chronic lymphocytic leukemia (B-CLL). Further, miR-10 is apossible regulator of the translation of Hox Genes, particularly Hox 3and Hox 4 (or Scr and Dfd in Drosophila).

In general, the claimed nucleic acid molecules may be used as amodulator of the expression of genes which are at least partiallycomplementary to said nucleic acid. Further, miRNA molecules may act astarget for therapeutic screening procedures, e.g. inhibition oractivation of miRNA molecules might modulate a cellular differentiationprocess, e.g. apoptosis.

Furthermore, existing miRNA molecules may be used as starting materialsfor the manufacture of sequence-modified miRNA molecules, in order tomodify the target-specificity thereof, e.g. an oncogene, amultidrug-resistance gene or another therapeutic target gene. The novelengineered miRNA molecules preferably have an identity of at least 80%to the starting miRNA, e.g. as depicted in Tables 1, 2, 3 and 4.Further, miRNA molecules can be modified, in order that they aresymetrically processed and then generated as double-stranded siRNAswhich are again directed against therapeutically relevant targets.

Furthermore, miRNA molecules may be used for tissue reprogrammingprocedures, e.g. a differentiated cell line might be transformed byexpression of miRNA molecules into a different cell type or a stem cell.

For diagnostic or therapeutic applications, the claimed RNA moleculesare preferably provided as a pharmaceutical composition. Thispharmaceutical composition comprises as an active agent at least onenucleic acid molecule as described above and optionally apharmaceutically acceptable carrier.

The administration of the pharmaceutical composition may be carried outby known methods, wherein a nucleic acid is introduced into a desiredtarget cell in vitro or in vivo.

Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods [30,31, 32, 33, 34]. A recent addition to this arsenal of techniques for theintroduction of DNA into cells is the use of cationic liposomes [35].

Commercially available cationic lipid formulations are e.g. Tfx 50(Promega) or Lipofectamin 2000 (Life Technologies).

The composition may be in form of a solution, e.g. an injectablesolution, a cream, ointment, tablet, suspension or the like. Thecomposition may be administered in any suitable way, e.g. by injection,by oral, topical, nasal, rectal application etc. The carrier may be anysuitable pharmaceutical carrier. Preferably, a carrier is used, which iscapable of increasing the efficacy of the RNA molecules to enter thetarget-cells. Suitable examples of such carriers are liposomes,particularly cationic liposomes.

Further, the invention relates to a method of identifying novelmicroRNA-molecules and precursors thereof, in eukaryotes, particularlyin vertebrates and more particularly in mammals, such as humans or mice.This method comprises: ligating 5′- and 3′-adapter-molecules to the endof a size-fractionated RNA-population, reverse transcribing saidadapter-ligated RNA-population, and characterizing said reversetranscribed RNA-molecules, e.g. by amplification, concatamerization,cloning and sequencing.

A method as described above already has been described in (8), however,for the identification of siRNA molecules. Surprisingly, it was foundnow that the method is also suitable for identifying the miRNA moleculesor precursors thereof as claimed in the present application.

Further, it should be noted that as 3′-adaptor for derivatization of the3′-OH group not only 4-hydroxymethylbenzyl but other types ofderivatization groups, such as alkyl, alkyl amino, ethylene glycol or3′-deoxy groups are suitable.

Further, the invention shall be explained in more detail by thefollowing Figures and Examples:

FIGURE LEGENDS

FIG. 1A. Expression of D. melanogaster miRNAs. Northern blots of totalRNA isolated from staged populations of D. melanogaster were probed forthe indicated miRNAs. The position of 76-nt val-tRNA is also indicatedon the blots. 5S rRNA serves as loading control. E, embryo; L, larvalstage; P, pupae; A, adult; S2, Schneider-2 cells. It should be pointedout, that S2 cells are polyclonal, derived from an unknown subset ofembryonic tissues, and may have also lost some features of their tissueof origin while maintained in culture. miR-3 to miR-6 RNAs were notdetectable in S2 cells (data not shown). miR-14 was not detected byNorthern blotting and may be very weakly expressed, which is consistentwith its cloning frequency. Similar miRNA sequences are difficult todistinguish by Northern blotting because of potentialcross-hybridization of probes.

FIG. 1B. Expression of vertebrate miRNAs. Northern blots of total RNAisolated from HeLa cells, mouse kidneys, adult zebrafish, frog ovaries,and S2 cells were probed for the indicated miRNAs. The position of 76-ntval-tRNA is also indicated on the blots. 5S rRNA from the preparationsof total RNA from the indicated species is also shown. The gels used forprobing of miR-18, miR-19a, miR-30, and miR-31 were not run as far asthe other gels (see tRNA marker position). miR-32 and miR-33 were notdetected by Northern blotting, which is consistent with their lowcloning frequency. Oligodeoxynucleotides used as Northern probes were:

let-7a, 5′ TACTATACAACCTACTACCTCAATTTGCC; (SEQ ID NO:1) let-7d,5′ ACTATGCAACCTACTACCTCT; (SEQ ID NO:2) let-7e,5′ ACTATACAACCTCCTACCTCA; (SEQ ID NO:3) D. melanogaster val-tRNA,5′ TGGTGTTTCCGCCCGGGAA; (SEQ ID NO:4) miR-1, 5′ TGGAATGTAAAGAAGTATGGAG;(SEQ ID NO:5) miR-2b, 5′ GCTCCTCAAAGCTGGCTGTGATA; (SEQ ID NO:6) miR-3,5′ TGAGACACACTTTGCCCAGTGA; (SEQ ID NO:7) miR-4,5′ TCAATGGTTGTCTAGCTTTAT; (SEQ ID NO:8) miR-5,5′ CATATCACAACGATCGTTCCTTT; (SEQ ID NO:9) miR-6,5′ AAAAAGAACAGCCACTGTGATA; (SEQ ID NO:10) miR-7,5′ TGGAAGACTAGTGATTTTGTTGT; (SEQ ID NO:11) miR-8,5′ GACATCTTTACCTGACAGTATTA; (SEQ ID NO:12) miR-9,5′ TCATACAGCTAGATAACCAAAGA; (SEQ ID NO:13) miR-10,5′ ACAAATTCGGATCTACAGGGT; (SEQ ID NO:14) miR-11,5′ GCAAGAACTCAGACTGTGATG; (SEQ ID NO:15) miR-12,5′ ACCAGTACCTGATGTAATACTCA; (SEQ ID NO:16) miR-13a,5′ ACTCGTCAAAATGGCTGTGATA; (SEQ ID NO:17) miR-14,5′ TAGGAGAGAGAAAAAGACTGA; (SEQ ID NO:18) miR-15,5′ TAGCAGCACATAATGGTTTGT; (SEQ ID NO:19) miR-16,5′ GCCAATATTTACGTGCTGCTA; (SEQ ID NO:20) miR-17,5′ TACAAGTGCCTTCACTGCAGTA; (SEQ ID NO:21) miR-18,5′ TATCTGCACTAGATGCACCTTA; (SEQ ID NO:22) miR-19a,5′ TCAGTTTTGCATAGATTTGCACA; (SEQ ID NO:23) miR-20,5′ TACCTGCACTATAAGCACTTTA; (SEQ ID NO:24) miR-21,5′ TCAACATCAGTCTGATAAGCTA; (SEQ ID NO:25) miR-22,5′ ACAGTTCTTCAACTGGCAGCTT; (SEQ ID NO:26) miR-23,5′ GGAAATCCCTGGCAATGTGAT; (SEQ ID NO:27) miR-24,5′ CTGTTCCTGCTGAACTGAGCCA; (SEQ ID NO:28) miR-25,5′ TCAGACCGAGACAAGTGCAATG; (SEQ ID NO:29) miR-26a,5′ AGCCTATCCTGGATTACTTGAA; (SEQ ID NO:30) miR-27;5′ AGCGGAACTTAGCCACTGTGAA; (SEQ ID NO:31) miR-28,5′ CTCAATAGACTGTGAGCTCCTT; (SEQ ID NO:32) miR-29,5′ AACCGATTTCAGATGGTGCTAG; (SEQ ID NO:33) miR-30,5′ GCTGCAAACATCCGACTGAAAG; (SEQ ID NO:34) miR-31,5′ CAGCTATGCCAGCATCTTGCCT; (SEQ ID NO:35) miR-32,5′ GCAACTTAGTAATGTGCAATA; (SEQ ID NO:36) miR-33,5′ TGCAATGCAACTACAATGCACC. (SEQ ID NO:37)

FIG. 2. Genomic organization of miRNA gene clusters. The precursorstructure is indicated as box and the location of the miRNA within theprecursor is shown in gray; the chromosomal location is also indicatedto the right. (A) D. melanogaster miRNA gene clusters. (B) Human miRNAgene clusters. The cluster of let-7a-1 and let-7f-1 is separated by26500nt from a copy of let-7d on chromosome 9 and 17. A cluster oflet-7a-3 and let-7b, separated by 938 nt on chromosome 22, is notillustrated.

FIG. 3. Predicted precursor structures of D. melanogaster miRNAs. RNAsecondary structure prediction was performed using mfold version 3.1[28]and manually refined to accommodate G/U wobble base pairs in thehelical segments. The miRNA sequence is underlined. The actual size ofthe stem-loop structure is not known experimentally and may be slightlyshorter or longer than represented. Multicopy miRNAs and theircorresponding precursor structures are also shown.

FIG. 4. Predicted precursor structures of human miRNAs. For legend, seeFIG. 3.

FIG. 5. Expression of novel mouse miRNAs. Northern blot analysis ofnovel mouse miRNAs. Total RNA from different mouse tissues was blottedand probed with a 5′-radiolabeled oligodeoxynucleotide complementary tothe indicated miRNA. Equal loading of total RNA on the gel was verifiedby ethidium bromide staining prior to transfer; the band representingtRNAs is shown. The fold-back precursors are indicated with capital L.Mouse brains were dissected into midbrain, mb, cortex, cx, cerebellum,cb. The rest of the brain, rb, was also used. Other tissues were heart,ht, lung, lg, liver, lv, colon, co, small intestine, si, pancreas, pc,spleen, sp, kidney, kd, skeletal muscle, sm, stomach, st, H, human HelaSS3 cells. Oligodeoxynucleotides used as Northern probes were:

miR-1a, CTCCATACTTCTTTACATTCCA; (SEQ ID NO:38) miR-30b,GCTGAGTGTAGGATGTTTACA; (SEQ ID NO:39) miR-30a-s,GCTTCCAGTCGAGGATGTTTACA; (SEQ ID NO:40) miR-99b, CGCAAGGTCGGTTCTACGGGTG;(SEQ ID NO:41) miR-101, TCAGTTATCACAGTACTGTA; (SEQ ID NO:42) miR-122a,ACAAACACCATTGTCACACTCCA; (SEQ ID NO:43) miR-124a, TGGCATTCACCGCGTGCCTTA;(SEQ ID NO:44) miR-125a, CACAGGTTAAAGGGTCTCAGGGA; (SEQ ID NO:45)miR-125b, TCACAAGTTAGGGTCTCAGGGA; (SEQ ID NO:46) miR-127,AGCCAAGCTCAGACGGATCCGA; (SEQ ID NO:47) miR-128, AAAAGAGACCGGTTCACTCTGA;(SEQ ID NO:48) miR-129, GCAAGCCCAGACCGAAAAAAG; (SEQ ID NO:49) miR-130,GCCCTTTTAACATTGCACTC; (SEQ ID NO:50) miR-131, ACTTTCGGTTATCTAGCTTTA;(SEQ ID NO:51) miR-132, ACGACCATGGCTGTAGACTGTTA; (SEQ ID NO:52) miR-143,TGAGCTACAGTGCTTCATCTCA. (SEQ ID NO:53)

FIG. 6. Potential orthologs of lin-4 stRNA. (A) Sequence alignment of C.elegans lin-4 stRNA [SEQ ID NO: 560] with mouse miR-125a [SEQ ID NO:562] and miR-125b [SEQ ID NO: 152] and the D. melanogaster miR-125 [SEQID NO: 561]. Differences are highlighted by gray boxes. (B) Northernblot of total RNA isolated from staged populations of D. melanogaster,probed for miR-125. E, embryo; L, larval stage; P, pupae; A, adult; S2,Schneider-2 cells.

FIG. 7. Predicted precursor structures of miRNAs, sequence accessionnumbers and homology information. RNA secondary structure prediction wasperformed using mfold version 3.1 and manually refined to accommodateG/U wobble base pairs in the helical segments. Dashes were inserted intothe secondary structure presentation when asymmetrically bulgednucleotides had to be accommodated. The excised miRNA sequence isunderlined. The actual size of the stem-loop structure is not knownexperimentally and may be slightly shorter or longer than represented.Multicopy miRNAs and their corresponding precursor structures are alsoshown. In cases where no mouse precursors were yet deposited in thedatabase, the human orthologs are indicated. miRNAs which correspond toD. melanogaster or human sequences are included. Published C. elegansmiRNAs [36, 37] are also included in the table. A recent set of new HeLacell miRNAs is also indicated [46]. If several ESTs were retrieved forone organism in the database, only those with different precursorsequences are listed. miRNA homologs found in other species areindicated. Chromosomal location and sequence accession numbers, andclusters of miRNA genes are indicated. Sequences from cloned miRNAs weresearched against mouse and human in GenBank (including trace data), andagainst Fugu rubripes and Danio rerio at www.jgi.doe.gov andwww.sanger.ac.uk, respectively.

EXAMPLE 1

MicroRNAs from D. melanogaster and Human.

We previously developed a directional cloning procedure to isolatesiRNAs after processing of long dsRNAs in Drosophila melanogaster embryolysate (8). Briefly, 5′ and 3′ adapter molecules were ligated to theends of a size-fractionated RNA population, followed by reversetranscription, PCR amplification, concatamerization, cloning andsequencing. This method, originally intended to isolate siRNAs, led tothe simultaneous identification of 14 novel 20- to 23-nt short RNAswhich are encoded in the D. melanogaster genome and which are expressedin 0 to 2 h embryos (Table 1). The method was adapted to clone RNAs in asimilar size range from HeLa cell total RNA (14), which led to theidentification of 19 novel human stRNAs (Table 2), thus providingfurther evidence for the existence of a large class of small RNAs withpotential regulatory roles. According to their small size, we refer tothese novel RNAs as microRNAs or miRNAs. The miRNAs are abbreviated asmiR-1 to miR-33, and the genes encoding miRNAs are named mir-1 tomir-33. Highly homologous miRNAs are classified by adding a lowercaseletter, followed by a dash and a number for designating multiple genomiccopies of a mir gene.

The expression and size of the cloned, endogenous short RNAs was alsoexamined by Northern blotting (FIG. 1, Table 1 and 2). Total RNAisolation was performed by acid guanidiniumthiocyanate-phenol-chloroform extraction [45]. Northern analysis wasperformed as described [1], except that the total RNA was resolved on a15% denaturing polyacrylamide gel, transferred onto Hybond-N+membrane(Amersham Pharmacia Biotech), and the hybridization and wash steps wereperformed at 50° C. Oligodeoxynucleotides used as Northern probes were5′-32P-phosphorylated, complementary to the miRNA sequence and. 20 to 25nt in length.

5S rRNA was detected by ethidium staining of polyacrylamide gels priorto transfer. Blots were stripped by boiling in 0.1% aqueous sodiumdodecylsulfate/0.1×SSC (15 mM sodium chloride, 1.5 mM sodium citrate, pH7.0) for 10 min, and were re-probed up to 4 times until the 21-ntsignals became too weak for detection. Finally, blots were probed forval-tRNA as size marker.

For analysis of D. melanogaster RNAs, total RNA was prepared fromdifferent developmental stages, as well as cultured Schneider-2 (S2)cells, which originally derive from 20–24 h D. melanogaster embryos [15](FIG. 1, Table 1). miR-3 to miR-7 are expressed only duringembryogenesis and not at later developmental stages. The temporalexpression of miR-1, miR-2 and miR-8 to miR-13 was less restricted.These miRNAs were observed at all developmental stages thoughsignificant variations in the expression levels were sometimes observed.Interestingly, miR-1, miR-3 to miR-6, and miR-8 to miR-11 werecompletely absent from cultured Schneider-2 (S2) cells, which wereoriginally derived from 20–24 h D. melanogaster embryos [15], whilemiR-2, miR-7, miR-12, and miR-1.3 were present in S2 cells, thereforeindicating cell type-specific miRNA expression. miR-1, miR-8, and miR-12expression patterns are similar to those of lin-4 stRNA in C. elegans,as their expression is strongly upregulated in larvae and sustained toadulthood [16]. miR-9 and miR-11 are present at all stages but arestrongly reduced in the adult which may reflect a maternal contributionfrom germ cells or expression in one sex only.

The mir-3 to mir-6 genes are clustered (FIG. 2A), and mir-6 is presentas triple repeat with slight variations in the mir-6 precursor sequencebut not in the miRNA sequence itself. The expression profiles of miR-3to miR-6 are highly similar (Table 1); which suggests that a singleembryo-specific precursor transcript may give rise to the differentmiRNAs, or that the same enhancer regulates miRNA-specific promoters.Several other fly miRNAs are also found in gene clusters (FIG. 2A).

The expression of HeLa cell miR-15 to miR-33 was examined by Northernblotting using HeLa cell total RNA, in addition to total RNA preparedfrom mouse kidneys, adult zebrafish, Xenopus laevis ovary, and D.melanogaster S2 cells (FIG. 1B, Table 2). miR-15 and miR-16 are encodedin a gene cluster (FIG. 2B) and are detected in mouse kidney, fish, andvery weakly in frog ovary, which may result from miRNA expression insomatic ovary tissue rather than oocytes. mir-17 to mir-20 are alsoclustered (FIG. 2B), and are expressed in HeLa cells and fish, butundetectable in mouse kidney and frog ovary (FIG. 1, Table 2), andtherefore represent a likely case of tissue-specific miRNA expression.

The majority of vertebrate and invertebrate miRNAs identified in thisstudy are not related by sequence, but a few exceptions, similar to thehighly conserved let-7 RNA [6], do exist. Sequence analysis of the D.melanogaster miRNAs revealed four such examples of sequence conservationbetween invertebrates and vertebrates. miR-1 homologs are encoded in thegenomes of C. elegans, C. briggsae, and humans, and are found in cDNAsfrom zebrafish, mouse, cow and human. The expression of mir-1 wasdetected by Northern blotting in total RNA from adult zebrafish and C.elegans, but not in total RNA from HeLa cells or mouse kidney (Table 2and data not shown). Interestingly, while mir-1 and let-7 are expressedboth in adult flies (FIG. 1A) [6] and are both undetected in S2 cells,miR-1 is, in contrast to let-7, undetectable in HeLa cells. Thisrepresents another case of tissue-specific expression of a miRNA, andindicates that miRNAs may not only play a regulatory role indevelopmental timing, but also in tissue specification. miR-7 homologswere found by database searches in mouse and human genomic and expressedsequence tag sequences (ESTs). Two mammalian miR-7 variants arepredicted by sequence analysis in mouse and human, and were detected byNorthern blotting in HeLa cells and fish, but not in mouse kidney (Table2). Similarly, we identified mouse and human miR-9 and miR-10 homologsby database searches but only detected mir-10 expression in mousekidney.

The identification of evolutionary related miRNAs, which have alreadyacquired multiple sequence mutations, was not possible by standardbioinformatic searches. Direct comparison of the D. melanogaster miRNAswith the human miRNAs identified an 11-nt segment shared between D.melanogaster miR-6 and HeLa miR-27, but no further relationships weredetected. One may speculate that most miRNAs only act on a single targetand therefore allow for rapid evolution by covariation, and that highlyconserved miRNAs act on more than one target sequence, and thereforehave a reduced probability for evolutionary drift by covariation [6]. Analternative interpretation is that the sets of miRNAs from D.melanogaster and humans are fairly incomplete and that many more miRNAsremain to be discovered, which will provide the missing evolutionarylinks.

lin-4 and let-7 stRNAs were predicted to be excised from longertranscripts that contain approximately 30 base-pair stem-loop structures[1, 6]. Database searches for newly identified miRNAs revealed that allmiRNAs are flanked by sequences that have the potential to form stablestem-loop structures (FIGS. 3 and 4). In many cases, we were able todetect the predicted, approximately 70-nt precursors by Northernblotting (FIG. 1). Some miRNA precursor sequences were also identifiedin mammalian cDNA (EST) databases [27], indicating that primarytranscripts longer than 70-nt stem-loop precursors do also exist. Wenever cloned a 22-nt RNA complementary to any of the newly identifiedmiRNAs, and it is as yet unknown how the cellular processing machinerydistinguishes between the miRNA and its complementary strand.Comparative analysis of the precursor stem-loop structures indicatesthat the loops adjacent to the base-paired miRNA segment can be locatedon either side of the miRNA sequence (FIGS. 3 and 4), suggesting thatthe 5′ or 3′ location of the stem-closing loop is not the determinant ofmiRNA excision. It is also unlikely that the structure, length orstability of the precursor stem is the critical determinant as thebase-paired structures are frequently imperfect and interspersed by lessstable, non-Watson-Crick base pairs such as G/A, U/U, C/U, A/A, and G/Uwobbles. Therefore, a sequence-specific recognition process is a likelydeterminant for miRNA excision, perhaps mediated by members of theArgonaute (rde-1/ago1/piwi) protein family. Two members of this family,alg-1 and alg-2, have recently been shown to be critical for stRNAprocessing in C. elegans [13]. Members of the Argonaute protein familyare also involved in RNAi and PTGS. In D. melanogaster, these includeargonaute2, a component of the siRNA-endonuclease complex (RISC) [17],and its relative aubergine, which is important for silencing of repeatgenes [18]. In other species, these include rde-1, argonaute1, andqde-2, in C. elegans [19], Arabidopsis thaliana [20], and Neurosporacrassa [21], respectively. The Argonaute protein family thereforerepresents, besides the RNase III Dicer [12, 13], another evolutionarylink between RNAi and miRNA maturation.

Despite advanced genome projects, computer-assisted detection of genesencoding functional RNAs remains problematic [22]. Cloning of expressed,short functional RNAs, similar to EST approaches (RNomics), is apowerful alternative and probably the most efficient method foridentification of such novel gene products [23–26]. The number offunctional RNAs has been widely underestimated and is expected to growrapidly because of the development of new functional RNA cloningmethodologies.

The challenge for the future is to define the function and the potentialtargets of these novel miRNAs by using bioinformatics as-well asgenetics, and to establish a complete catalogue of time- andtissue-specific distribution of the already identified and yet to beuncovered miRNAs. lin-4 and let-7 stRNAs negatively regulate theexpression of proteins encoded by mRNAs whose 3′ untranslated regionscontain sites of complementarity to the stRNA [3–5].

Thus, a series of 33 novel genes, coding for 19- to 23-nucleotidemicroRNAs (miRNAs), has been cloned from fly embryos and human cells.Some of these miRNAs are highly conserved between vertebrates andinvertebrates and are developmentally or tissue-specifically expressed.Two of the characterized human miRNAs may function as tumor suppressorsin B-cell chronic lymphocytic leukemia. miRNAs are related to a smallclass of previously described 21- and 22-nt RNAs (lin-4 and let-7 RNAs),so-called small temporal RNAs (stRNAs), and regulate developmentaltiming in C. elegans and other species. Similar to stRNAs, miRNAs arepresumed to regulate translation of specific target mRNAs by binding topartially complementary sites, which are present in their3′-untranslated regions.

Deregulation of miRNA expression may be a cause of human disease, anddetection of expression of miRNAs may become useful as a diagnostic.Regulated expression of miRNAs in cells or tissue devoid of particularmiRNAs may be useful for tissue engineering, and delivery or transgenicexpression of miRNAs may be useful for therapeutic intervention. miRNAsmay also represent valuable drug targets itself. Finally, miRNAs andtheir precursor sequences may be engineered to recognize therapeuticvaluable targets.

EXAMPLE 2

miRNAs from Mouse.

To gain more detailed insights into the distribution and function ofmiRNAs in mammals, we investigated the tissue-specific distribution ofmiRNAs in adult mouse. Cloning of miRNAs from specific tissues waspreferred over whole organism-based cloning because low-abundance miRNAsthat normally go undetected by Northern blot analysis are identifiedclonally. Also, in situ hybridization techniques for detecting 21-ntRNAs have not yet been developed. Therefore, 19- to 25-nucleotide RNAswere cloned and sequenced from total RNA, which was isolated from 18.5weeks old BL6 mice. Cloning of miRNAs was performed as follows: 0.2 to 1mg of total RNA was separated on a 15% denaturing polyacrylamide gel andRNA of 19- to 25-nt size was recovered. A 5′-phosphorylated 3′-adapterolignonucleotide (5′-pUUUaaccgcgaattccagx: uppercase, RNA; lowercase,DNA; p, phosphate; x, 3′-Amino-Modifier C-7, ChemGenes, Ashland, Mass.,USA, Cat. No. NSS-1004; SEQ ID NO: 54) and a 5′-adapter oligonucleotide(5′-acggaattcctcactAAA: uppercase, RNA; lowercase, DNA; SEQ ID NO: 55)were ligated to the short RNAs. RT/PCR was performed with 3′-primer(5-GACTAGCTGGAATTCGCGGTTAAA; SEQ ID NO: 558) and 5′-primer(5′-CAGCCAACGGAATTCCTCACTAAA; SEQ ID NO: 57). In order to introduce BanI restriction sites, a second PCR was performed using the primer pair5-CAGCCAACAGGCACCGAATTCCTCACTAAA (SEQ ID NO: 559) and5′-GACTAGCTTGGTGCCGAATTCGCGGTTAAA (SEQ ID NO: 56), followed byconcatamerization after Ban I digestion and T4 DNA ligation. Concatamersof 400 to 600 basepairs were cut out from 1.5% agarose gels andrecovered by Biotrap (Schleicher & Schuell) electroelution (1×TAEbuffer) and by ethanol precipitation. Subsequently, the 3′ ends of theconcatamers were filled in by incubating for 15 min at 72° C. with Taqpolymerase in standard PCR reaction mixture. This solution was diluted3-fold with water and directly used for ligation into pCR2.1 TOPOvectors. Clones were screened for inserts by PCR and 30 to 50 sampleswere subjected to sequencing. Because RNA was prepared from combiningtissues of several mice, minor sequence variations that were detectedmultiple times in multiple clones may reflect polymorphisms rather thanRT/PCR mutations. Public database searching was used to identify thegenomic sequences encoding the approx. 21-nt RNAs. The occurrence of a20 to 30 basepair fold-back structure involving the immediate upstreamor downstream flanking sequences was used to assign miRNAs [36–38].

We examined 9 different mouse tissues and identified 34 novel miRNAs,some of which are highly tissue-specifically expressed (Table 3 and FIG.5). Furthermore, we identified 33 new miRNAs from different mousetissues and also from human Soas-2 osteosarcoma cells (Table 4). miR-1was previously shown by Northern analysis to be strongly expressed inadult heart, but not in brain, liver, kidney, lung or colon [37]. Herewe show that miR-1 accounts for 45% of all mouse miRNAs found in heart,yet miR-1 was still expressed at a low level in liver and midbrain eventhough it remained undetectable by Northern analysis. Three copies orpolymorphic alleles of miR-1 were found in mice. The conservation oftissue-specific miR-1 expression between mouse and human providesadditional evidence for a conserved regulatory role of this miRNA. Inliver, variants of miR-122 account for 72% of all cloned miRNAs andmiR-122 was undetected in all other tissues analyzed. In spleen, miR-143appeared to be most abundant, at a frequency of approx. 30%. In colon,miR-142-as, was cloned several times and also appeared at a frequency of30%. In small intestine, too few miRNA sequences were obtained to permitstatistical analysis. This was due to strong RNase activity in thistissue, which caused significant breakdown of abundant non-coding RNAs,e.g. rRNA, so that the fraction of miRNA in the cloned sequences wasvery low. For the same reason, no miRNA sequences were obtained frompancreas.

To gain insights in neural tissue miRNA distribution, we analyzedcortex, cerebellum and midbrain. Similar to heart, liver and smallintestine, variants of a particular miRNA, miR-124, dominated andaccounted for 25 to 48% of all brain miRNAs. miR-101, -127, -128, -131,and -132, also cloned from brain tissues, were further analyzed byNorthern blotting and shown to be predominantly brain-specific. Northernblot analysis was performed as described in Example 1. tRNAs and 5S rRNAwere detected by ethidium staining of polyacrylamide gels prior totransfer to verify equal loading. Blots were stripped by boiling indeionized water for 5 min, and reprobed up to 4 times until the 21-ntsignals became too weak for detection.

miR-125a and miR-125b are very similar to the sequence of C. eleganslin-4 stRNA and may represent its orthologs (FIG. 6A). This is of greatinterest because, unlike let-7 that was readily detected in otherspecies, lin-4 has acquired a few mutations in the central region andthus escaped bioinformatic database searches. Using the mouse sequencemiR-125b, we could readily identify its ortholog in the D. melanogastergenome. miR-125a and miR-125b differ only by a central diuridineinsertion and a U to C change. miR-125b is very similar to lin-4 stRNAwith the differences located only in the central region, which ispresumed to be bulged out during target mRNA recognition [41]. miR-125aand miR-125b were cloned from brain tissue, but expression was alsodetected by Northern analysis in other tissues, consistent with the rolefor lin-4 in regulating neuronal remodeling by controlling lin-14expression [43]. Unfortunately, prthologs to C. elegans lin-14 have notbeen described and miR-125 targets remain to be identified in D.melanogaster or mammals. Finally, miR-125b expression is alsodevelopmentally regulated and only detectable in pupae and adult but notin embryo or larvae of D. melanogaster (FIG. 6B).

Sequence comparison of mouse miRNAs with previously described miRNAreveals that miR-99b and miR-99a are similar to D. melanogaster, mouseand human miR-10 as well as C. elegans miR-51 [36], miR-141 is similarto D. melanogaster miR-8, miR-29b is similar to C. elegans miR-83, andmiR-131 and miR-142-s are similar to D. melanogaster miR-4 and C.elegans miR-79 [36]. miR-124a is conserved between invertebrates andvertebrates. In this respect it should be noted that for almost everymiRNA cloned from mouse was also encoded in the human genome, andfrequently detected in other vertebrates, such as the pufferfish, Fugurubripes, and the zebrafish, Danio rerio. Sequence conservation maypoint to conservation in function of these miRNAs. Comprehensiveinformation about orthologous sequences is listed in FIG. 7.

In two cases both strands of miRNA precursors were cloned (Table 3),which was previously observed once for a C. elegans miRNA [36]. It isthought that the most frequently cloned strand of a miRNA precursorrepresents the functional miRNA, which is miR-30c-s and miRi142-as, sand as indicating the 5′ or 3′ side of the fold-back structure,respectively.

The mir-142 gene is located on chromosome 17, but was also found at thebreakpoint junction of a t(8; 17) translocation, which causes anaggressive B-cell leukemia due to strong up-regulation of a translocatedMYC gene [44]. The translocated MYC gene, which was also truncated atthe first exon, was located only 4-nt downstream of the 3′-end of themiR-142 precursor. This suggests that translocated MYC was under thecontrol of the upstream miR-142 promoter. Alignment of mouse and humanmiR-142 containing EST sequences indicate an approximately 20 ntconserved sequence element downstream of the mir-142 hairpin. Thiselement was lost in the translocation. It is conceivable that theabsence of the conserved downstream sequence element in the putativemiR-142/mRNA fusion prevented the recognition of the transcript as amiRNA precursor and therefore may have caused accumulation of fusiontranscripts and overexpression of MYC.

miR-155, which was cloned from colon, is excised from the knownnoncoding BIC RNA [47]. BIC was originally identified as a genetranscriptionally activated by promoter insertion at a common retroviralintegration site in B cell lymphomas induced by avian leukosis virus.Comparison of BIC cDNAs from human, mouse and chicken revealed 78%identity over 138 nucleotides [47]. The identity region covers themiR-155 fold-back precursor and a few conserved boxes downstream of thefold-back sequence. The relatively high level of expression of BIC inlymphoid organs and cells in human, mouse and chicken implies anevolutionary conserved function, but BIC RNA has also been detected atlow levels in non-hematopoietic tissues [47].

Another interesting observation was that segments of perfectcomplementarity to miRNAs are not observed in mRNA sequences or ingenomic sequences outside the miRNA inverted repeat. Although this couldbe fortuitous, based on the link between RNAi and miRNA processing [11,13, 43] it may be speculated that miRNAs retain the potential to cleaveperfectly complementary target RNAs. Because translational controlwithout target degradation could provide more flexibility it may bepreferred over mRNA degradation.

In summary, 63 novel miRNAs were identified from mouse and 4 novelmiRNAs were identified from human Soas-2 osteosarcoma cells (Table 3 andTable 4), which are conserved in human and often also in othernon-mammalian vertebrates. A few of these miRNAs appear to be extremelytissue-specific, suggesting a critical role for some miRNAs intissue-specification and cell lineage decisions. We may have alsoidentified the fruitfly and mammalian ortholog of C. elegans lin-4stRNA. The establishment of a comprehensive list of miRNA sequences willbe instrumental for bioinformatic approaches that make use of completedgenomes and the power of phylogenetic comparison in order to identifymiRNA-regulated target mRNAs.

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TABLE 1 D. melanogaster miRNAs. The sequences given represent the mostabundant, and typically longest miRNA sequence identified by cloning;miRNAs frequently vary in length by one or two nucleotides at their 3′termini. From 222 short RNAs sequenced, 69 (31%) corresponded to miRNAs,103 (46%) to already characterized functional RNAs (rRNA, 7SL RNA,tRNAs), 30 (14%) to transposon RNA fragments, and 20 (10%) sequenceswith no database entry. The frequency (freq.) for cloning a particularmiRNA relative to all identified miRNAs is indicated in percent. Resultsof Northern blotting of total RNA isolated from staged populations of D.melanogaster are summarized. E, embryo; L, larval stage; P, pupae; A,adult; S2, Schneider-2 cells. The strength of the signal within eachblot is represented from strongest (+ + +) to undetected (−). let-7stRNA was probed as control. Genbank accession numbers and homologs ofmiRNAs identified by database searching in other species are provided assupplementary material. freq. E E L1 + miRNA sequence (5′ to 3′) (%) 0–3h 0–6 h L2 L3 P A S2 miR-1 UGGAAUGUAAAGAAGUAUGGAG (SEQ ID NO:58) 32 + ++ + + + + + + + + + + − miR-2a* UAUCAGCCAGCUUUGAUGAGC (SEQ ID NO:59) 3miR-2b* UAUCACAGCCAGCUUUGAGGAGC (SEQ ID NO:60) 3 + + + + + + + + + + + ++ + + miR-3 UCACUGGGCAAAGUGUGUCUCA# 9 + + + + + + − − − − − miR-4AUAAAGCUAGACAACCAUUGA (SEQ ID NO:62) 6 + + + + + + − − − − − miR-5AAAGGAACGAUCGUUGUGAUAUG (SEQ ID NO:63) 1 + + + + + + +/− +/− − − − miR-6UAUCACAGUGGCUGUUCUUUUU (SEQ ID NO:64) 13 + + + + + + +/− +/− − − − miR-7UGGAAGACUAGUGAUUUUGUUGU (SEQ ID NO:65) 4 + + + + + +/− +/− +/− +/− +/−miR-8 UAAUACUGUCAGGUAAAGAUGUC (SEQ ID NO:66) 3 +/− +/− + + + + + + ++ + + − miR-9 UCUUUGGUUAUCUAGCUGUAUGA (SEQ ID NO:67) 7 + + + + + + + ++ + + + + + +/− − miR-10 ACCCUGUAGAUCCGAAUUUGU (SEQ ID NO:68) 1 + + + ++ + + +/− + − miR-11 CAUCACAGUCUGAGUUCUUGC (SEQ ID NO:69) 7 + + + + + ++ + + + + + + + + + − miR-12 UGAGUAUUACAUCAGGUACUGGU (SEQ ID NO:70)7 + + + + + + + + + + +/− miR-13a* UAUCACAGCCAUUUUGACGAGU (SEQ ID NO:71)1 + + + + + + + + + + + + + + + + + + + miR-13b* UAUCACAGCCAUUUUGAUGAGU(SEQ ID NO:72) 0 miR-14 UCAGUCUUUUUCUCUCUCCUA (SEQ ID NO:73) 1 − − − − −− − let-7 UGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO:74) 0 − − − − + + + + + + −# = (SEQ ID NO:61) *Similar miRNA sequences are difficult to distinguishby Northern blotting because of potential cross-hybridization of probes.

TABLE 2 Human miRNAs. From 220 short RNAs sequenced, 100 (45%)corresponded to miRNAs, 53 (24%) to already characterized functionalRNAs (rRNA, snRNAs, tRNAs), and 67 (30%) sequences with no databaseentry. Results of Northern blotting of total RNA isolated from differentvertebrate species and S2 cells are indicated. For legend, see Table 1.freq. HeLa mouse adult frog miRNA sequence (5′ to 3′) (%) cells kidneyfish ovary S2 let-7a* UGAGGUAGUAGGUUGUAUAGUU# 10 + + + + + + + + + − −let-7b* UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:76) 13 let-7c*UGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO:77) 3 let-7d* AGAGGUAGUAGGUUGCAUAGU(SEQ ID NO:78) 2 + + + + + + + + + − − let-7e* UGAGGUAGGAGGUUGUAUAGU(SEQ ID NO:79) 2 + + + + + + + + + − − let-7f* UGAGGUAGUAGAUUGUAUAGUU(SEQ ID NO:80) 1 miR-15 UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO:81) 3 + + ++ + + +/− − miR-16 UAGCAGCACGUAAAUAUUGGCG (SEQ ID NO:82) 10 + + + + +/−+/− − miR-17 ACUGCAGUGAAGGCACUUGU (SEQ ID NO:83) 1 + + + − − − − miR-18UAAGGUGCAUCUAGUGCAGAUA (SEQ ID NO:84) 2 + + + − − − − miR-19a*UGUGCAAAUCUAUGCAAAACUGA (SEQ ID NO:85) 1 + + + − +/− − − miR-19b*UGUGCAAAUCCAUGCAAAACUGA (SEQ ID NO:86) 3 miR-20 UAAAGUGCUUAUAGUGCAGGUA(SEQ ID NO:87) 4 + + + − + − − miR-21 UAGCUUAUCAGACUGAUGUUGA (SEQ IDNO:88) 10 + + + + + + − − miR-22 AAGCUGCCAGUUGAAGAACUGU (SEQ ID NO:89)10 + + + + + + + +/− − miR-23 AUCACAUUGCCAGGGAUUUCC (SEQ ID NO:90) 2+ + + + + + + + + + − miR-24 UGGCUCAGUUCAGCAGGAACAG (SEQ ID NO:91) 4 + ++ + + + + − − miR-25 CAUUGCACUUGUCUCGGUCUGA (SEQ ID NO:92) 3 + + + + + +− − miR-26a* UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO:93) 2 + + + + + + − −miR-26b* UUCAAGUAAUUCAGGAUAGGUU (SEQ ID NO:94) 1 − miR-27UUCACAGUGGCUAAGUUCCGCU (SEQ ID NO:95) 2 + + + + + + + + − − miR-28AAGGAGCUCACAGUCUAUUGAG (SEQ ID NO:96) 2 + + + + + + − − − miR-29CUAGCACCAUCUGAAAUCGGUU (SEQ ID NO:97) 2 + + + + +/− − − miR-30CUUUCAGUCGGAUGUUUGCAGC (SEQ ID NO:98) 2 + + + + + + + + + − − miR-31GGCAAGAUGCUGGCAUAGCUG (SEQ ID NO:99) 2 + + + − − − − miR-32UAUUGCACAUUACUAAGUUGC (SEQ ID NO:100) 1 − − − − − miR-33GUGCAUUGUAGUUGCAUUG (SEQ ID NO:101) 1 − − − − − miR-1UGGAAUGUAAAGAAGUAUGGAG (SEQ ID NO:102) 0 − − + − − miR-7UGGAAGACUAGUGAUUUUGUUGU (SEQ ID NO:103) 0 + − +/− − +/− miR-9UCUUUGGUUAUCUAGCUGUAUGA (SEQ ID NO:104) 0 − − − − − miR-10ACCCUGUAGAUCCGAAUUUGU (SEQ ID NO:105) 0 − + − − − # = (SEQ ID NO:75)*Similar miRNA sequences are difficult to distinguish by Northernblotting because of potential cross-hybridization of probes.

TABLE 3 Mouse miRNAs. The sequences indicated represent the longestmiRNA sequences identified by cloning. The 3′-terminus of miRNAs isoften truncated by one or two nucleotides. miRNAs that are more than 85%identical in sequence (i.e. share 18 out of 21 nucleotides) or contain1- or 2-nucleotide internal deletions are referred to by the same genenumber followed by a lowercase letter. Minor sequence variations betweenrelated miRNAs are generally found near the ends of the miRNA sequenceand are thought to not compromise target. RNA recognition. Minorsequence variations may also represent A to G and C to U changes, whichare accommodated as G-U wobble base pairs during target, recognition.miRNAs with the suffix -s or -as indicate RNAs derived from either the5′- half or the 3′-half of a miRNA precursor. Mouse brains weredissected into midbrain, mb, cortex, cx, cerebellum, cb. The tissuesanalyzed were heart, ht; liver, lv; small intestine, si; colon, co;cortex, ct; cerebellum, cb; midbrain, mb. Number of clones miRNAsequence (5′ to 3′) ht lv sp si co cx cb mb let-7aUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO:106) 3 1 1 7 let-7bUGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:107) 1 1 2 5 let-7cUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO:108) 2 2 5 19 let-7dAGAGGUAGUAGGUUGCAUAGU (SEQ ID NO:109) 2 2 2 2 let-7eUGAGGUAGGAGGUUGUAUAGU (SEQ ID NO:110) 1 2 let-7f UGAGGUAGUAGAUUGUAUAGUU(SEQ ID NO:111) 2 3 3 let-7g UGAGGUAGUAGUUUGUACAGUA (SEQ ID NO:112) 1 12 let-7h UGAGGUAGUAGUGUGUACAGUU (SEQ ID NO:113) 1 1 let-7iUGAGGUAGUAGUUUGUGCU (SEQ ID NO:114) 1 1 miR-1b UGGAAUGUAAAGAAGUAUGUAA(SEQ ID NO:115) 4 2 1 miR-1c UGGAAUGUAAAGAAGUAUGUAC (SEQ ID NO:116) 7miR-1d UGGAAUGUAAAGAAGUAUGUAUU (SEQ ID NO:117) 16 1 miR-9UCUUUGGUUAUCUAGCUGUAUGA (SEQ ID NO:118) 3 4 4 miR-15aUAGCAGCACAUAAUGGUUUGUG (SEQ ID NO:119) 1 2 miR-15bUAGCAGCACAUCAUGGUUUACA (SEQ ID NO:120) 1 miR-16 UAGCAGCACGUAAAUAUUGGCG(SEQ ID NO:121) 1 1 2 1 2 3 miR-18 UAAGGUGCAUCUAGUGCAGAUA (SEQ IDNO:122) 1 miR-19b UGUGCAAAUCCAUGCAAAACUGA (SEQ ID NO:123) 1 miR-20UAAAGUGCUUAUAGUGCAGGUAG (SEQ ID NO:124) 1 miR-21 UAGCUUAUCAGACUGAUGUUGA(SEQ ID NO:125) 1 1 2 1 miR-22 AAGCUGCCAGUUGAAGAACUGU (SEQ ID NO:126) 21 1 1 2 miR-23a AUCACAUUGCCAGGGAUUUCC (SEQ ID NO:127) 1 miR-23bAUCACAUUGCCAGGGAUUACCAC (SEQ ID NO:128) 1 miR-24 UGGCUCAGUUCAGCAGGAACAG(SEQ ID NO:129) 1 1 1 1 miR-26a UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO:120) 32 miR-26b UUCAAGUAAUUCAGGAUAGGUU (SEQ ID NO:131) 2 4 1 miR-27aUUCACAGUGGCUAAGUUCCGCU (SEQ ID NO:132) 1 2 1 1 2 miR-27bUUCACAGUGGCUAAGUUCUG (SEQ ID NO:133) 1 miR-29a CUAGCACCAUCUGAAAUCGGUU(SEQ ID NO:134) 1 1 1 miR-29b/miR-102 UAGCACCAUUUGAAAUCAGUGUU (SEQ IDNO:135) 1 1 5 3 miR-29c/ UAGCACCAUUUGAAAUCGGUUA (SEQ ID NO:136) 1 3 1miR-30a-s/miR-97 UGUAAACAUCCUCGACUGGAAGC (SEQ ID NO:137) 1 1 1miR-30a-as^(a) CUUUCAGUCGGAUGUUUGCAGC (SEQ ID NO:138) 1 miR-30bUGUAAACAUCCUACACUCAGC (SEQ ID NO:139) 1 2 miR-30cUGUAAACAUCCUACACUCUCAGC (SEQ ID NO:140) 2 1 1 miR-30dUGUAAACAUCCCCGACUGGAAG (SEQ ID NO:141) 1 miR-99a/miR-99ACCCGUAGAUCCGAUCUUGU (SEQ ID NO:142) 1 miR-99b CACCCGUAGAACCGACCUUGCG(SEQ ID NO:143) 1 miR-101 UACAGUACUGUGAUAACUGA (SEQ ID NO:144) 2 1 1miR-122a UGGAGUGUGACAAUGGUGUUUGU (SEQ ID NO:145) 3 miR-122bUGGAGUGUGACAAUGGUGUUUGA (SEQ ID NO:146) 11 miR-122a, bUGGAGUGUGACAAUGGUGUUUG (SEQ ID NO:147) 23 miR-123 CAUUAUUACUUUUGGUACGCG(SEQ ID NO:148) 1 2 miR-124a^(b) UUAAGGCACGCGG-UGAAUGCCA (SEQ ID NO:149)1 37 41 24 miR-124b UUAAGGCACGCGGGUGAAUGC (SEQ ID NO:150) 1 3 miR-125aUCCCUGAGACCCUUUAACCUGUG (SEQ ID NO:151) 1 1 miR-125bUCCCUGAGACCCU--AACUUGUGA (SEQ ID NO:152) 1 miR-126 UCGUACCGUGAGUAAUAAUGC(SEQ ID NO:153) 4 1 miR-127 UCGGAUCCGUCUGAGCUUGGCU (SEQ ID NO:154) 1miR-128 UCACAGUGAACCGGUCUCUUUU (SEQ ID NO:155) 2 2 2 miR-129CUUUUUUCGGUCUGGGCUUGC (SEQ ID NO:156) 1 miR-130 CAGUGCAAUGUUAAAAGGGC(SEQ ID NO:157) 1 miR-131 UAAAGCUAGAUAACCGAAAGU (SEQ ID NO:158) 1 1 1miR-132 UAACAGUCUACAGCCAUGGUCGU (SEQ ID NO:159) 1 miR-133UUGGUCCCCUUCAACCAGCUGU (SEQ ID NO:160) 4 1 miR-134UGUGACUGGUUGACCAGAGGGA (SEQ ID NO:161) 1 miR-135UAUGGCUUUUUAUUCCUAUGUGAA (SEQ ID NO:162) 1 miR-136ACUCCAUUUGUUUUGAUGAUGGA (SEQ ID NO:163) 1 miR-137 UAUUGCUUAAGAAUACGCGUAG(SEQ ID NO:164) 1 1 miR-138 AGCUGGUGUUGUGAAUC (SEQ ID NO:165) 1 miR-139UCUACAGUGCACGUGUCU (SEQ ID NO:166) 1 1 miR-140 AGUGGUGUUACCCUAUGGUAG(SEQ ID NO:167) 1 miR-141 AACACUGUCUGGUAAAGAUGG (SEQ ID NO:168) 1 1 1miR-142-s CAUAAAGUAGAAAGCACUAC (SEQ ID NO:169) 1 1 miR-142-as^(b)UGUAGUGUUUCCUACUUUAUGG (SEQ ID NO:170) 1 1 6 miR-143UGAGAUGAAGCACUGUAGCUCA (SEQ ID NO:171) 3 7 2 1 miR-144UACAGUAUAGAUGAUGUACUAG (SEQ ID NO:172) 2 1 miR-145GUCCAGUUUUCCCAGGAAUCCCUU (SEQ ID NO:173) 1 miR-146UGAGAACUGAAUUCCAUGGGUUU (SEQ ID NO:174) 1 miR-147 GUGUGUGGAAAUGCUUCUGCC(SEQ ID NO:175) 1 miR-148 UCAGUGCACUACAGAACUUUGU (SEQ ID NO:176) 1miR-149 UCUGGCUCCGUGUCUUCACUCC (SEQ ID NO:177) 1 miR-150UCUCCCAACCCUUGUACCAGUGU (SEQ ID NO:178) 1 miR-151 CUAGACUGAGGCUCCUUGAGGU(SEQ ID NO:179) 1 miR-152 UCAGUGCAUGACAGAACUUGG (SEQ ID NO:180) 1miR-153 UUGCAUAGUCACAAAAGUGA (SEQ ID NO:181) 1 miR-154UAGGUUAUCCGUGUUGCCUUCG (SEQ ID NO:182) 1 miR-155 UUAAUGCUAAUUGUGAUAGGGG(SEQ ID NO:183) 1 ^(a)The originally described miR-30 was renamed tomiR-30a-as in order to distinguish it from the miRNA derived from theopposite strand of the precursor encoded by the mir-30a gene. miR-30a-sis equivalent to miR-97 [46]. ^(b)A 1-nt length heterogeneity is foundon both 5′ and 3′ end. The 22-nt miR sequence is shown, but only 21-ntmiRNAs were cloned.

TABLE 4 Mouse and human miRNAs. The sequences indicated represent thelongest miRNA sequences identified by cloning. The 3′ terminus of miRNAsis often truncated by one or two nucleotides. miRNAs-that are more than85% identical in sequence (i.e. share 18 out of 21 nucleotides) orcontain 1- or 2-nucleotide internal deletions are referred to by thesame gene number followed by a lowercase letter. Minor sequencevariations between related miRNAs are generally found near the ends ofthe miRNA sequence and are thought to not compromise target RNArecognition. Minor sequence variations may also represent A to G and Cto U changes; which are accommodated as G-U wobble base pairs duringtarget recognition. Mouse brains were dissected into midbrain, mb,cortex, cx, cerebellum, cb. The tissues analyzed were lung, ln; liver,lv; spleen, sp; kidney, kd; skin, sk; testis, ts; ovary, ov; thymus,thy; eye, ey; cortex, ct; cerebellum, cb; midbrain, mb. The humanosteosarcoma cells SAOS-2 cells contained an inducible p53 gene (p53−,uninduced p53; p53+, induced p53); the differences in miRNAs identifiedfrom induced and uninduced SAOS cells were not statisticallysignificant. number of clones human SAOS- mouse tissues 2 cells miRNASequence (5′ to 3′) ln lv sp kd sk ts ov thy ey p53− p53+ miR-C1AACAUUCAACGCUGUCGGUGAGU 1 1 2 (SEQ ID NO.184) miR-C2UUUGGCAAUGGUAGAACUCACA 1 (SEQ ID NO.185) miR-C3 UAUGGCACUGGUAGAAUUCACUG1 (SEQ ID NO.186) miR-C4 CUUUUUGCGGUCUGGGCUUGUU 1 1 1 (SEQ ID NO.187)miR-C5 UGGACGGAGAACUGAUAAGGGU 2 (SEQ ID NO.188) miR-C6UGGAGAGAAAGGCAGUUC 1 (SEQ ID NO.189) miR-C7 CAAAGAAUUCUCCUUUUGGGCUU 1 1(SEQ ID NO.190) miR-C8 UCGUGUCUUGUGUUGCAGCCGG 1 (SEQ ID NO.191) miR-C9UAACACUGUCUGGUAACGAUG 1 (SEQ ID NO.192) miR-C10 CAUCCCUUGCAUGGUGGAGGGU 1(SEQ ID NO.193) miR-C11 GUGCCUACUGAGCUGACAUCAGU 1 (SEQ ID NO.194)miR-C12 UGAUAUGUUUGAUAUAUUAGGU 2 (SEQ ID NO.195) miR-C13CAACGGAAUCCCAAAAGCAGCU 2 1 (SEQ ID NO.196) miR-C14 CUGACCUAUGAAUUGACA 21 (SEQ ID NO.197) miR-C15 UACCACAGGGUAGAACCACGGA 1 (SEQ ID NO.198)miR-C16 AACUGGCCUACAAAGUCCCAG 1 (SEQ ID NO.199) miR-C17UGUAACAGCAACUCCAUGUGGA 1 (SEQ ID NO.200) miR-C18 UAGCAGCACAGAAAUAUUGGC 21 1 (SEQ ID NO.201) miR-C19 UAGGUAGUUUCAUGUUGUUGG 1 (SEQ ID NO.202)miR-C20 UUCACCACCUUCUCCACCCAGC 1 1 (SEQ ID NO.203) miR-C21GGUCCACAGGGGAGAUAGG 1 (SEQ ID NO.204) miR-C22 CCCAGUGUUCAGACUACCUGUU 1(SEQ ID NO.205) miR-C23 UAAUACUGCCUGGUAAUGAUGAC 2 1 (SEQ ID NO.206)miR-C24 UACUCAGUAAGGCAUUGUUCU 1 (SEQ ID NO.207) miR-C25AGAGGUAUAGCGCAUGGGAAGA 1 (SEQ ID NO.208) miR-C26 UGAAAUGUUUAGGACCACUAG 1(SEQ ID NO.209) miR-C27 UUCCCUUUGUCAUCCUAUGCCUG 1 (SEQ ID NO.210)miR-C28 UCCUUCAUUCCACCGGAGUCUG 1 (SEQ ID NO.211) miR-C29GUGAAAUGUUUAGGACCACUAGA 2 (SEQ ID NO.212) miR-C30 UGGAAUGUAAGGAAGUGUGUGG2 (SEQ ID NO.213) miR-C31 UACAGUAGUCUGCACAUUGGUU 1 (SEQ ID NO.214)miR-C32 CCCUGUAGAACCGAAUUUGUGU 1 1 (SEQ ID NO.215) miR-C33AACCCGUAGAUCCGAACUUGUGAA 1 (SEQ ID NO.216) miR-C34GCUUCUCCUGGCUCUCCUCCCUC 1 (SEQ ID NO.217)

TABLE 5 D. melanogaster miRNA sequences and genomic location. Thesequences given represent the most abundant, and typically longest miRNAsequences identified by cloning. It was frequently observed that miRNAsvary in length by one or two nucleotides at their 3′-terminus. From 222short RNAs sequenced; 69 (31%) corresponded to miRNAs, 103 (46%) toalready characterized functional RNAs (rRNA, 7SL RNA, tRNAs), 30 (14%)to transposon RNA fragments, and 20 (10%) sequences with no databaseentry. RNA sequences with a 5′- guanosine are likely to beunderrepresented due to the cloning procedure (8). miRNA homologs foundin other species are indicated. Chromosomal location (chr.) and GenBankaccession numbers (acc. nb.) are indicated. No ESTs matching miR-1 tomiR-14 were detectable by database searching. miRNA sequence (5′ to 3′)chr., acc. nb. remarks miR-1 UGGAAUGUAAAGAAGUAUGGAG (SEQ ID NO:58) 2L,AE003667 homologs: C. briggsae, G20U, AC87074; C. elegans G20U, U97405;mouse, G20U, G22U, AC020867; human, chr. 20, G20U, G22U, AL449263; ESTs:zebrafish, G20U, G22U, BF157- 601; cow, G20U, G22U, BE722- 224; human,G20U, G22U, AI220268 miR-2a UAUCACAGCCAGCUUUGAUGAGC (SEQ ID NO:59) 2L,AE003663 2 precursor variants clustered with a copy of mir-2b miR-2bUAUCACAGCCAGCUUUGAGGAGC (SEQ ID NO:60) 2L, AE003620 2 precursor variants2L, AE003663 miR-3 UCACUGGGCAAAGUGUGUCUCA (SEQ ID NO:61) 2R, AE003795 incluster mir-3 to mir-6 miR-4 AUAAAGCUAGACAACCAUUGA (SEQ ID NO:62) 2R,AE003795 in cluster mir-3 to mir-6 miR-5 AAAGGAACGAUCGUUGUGAUAUG (SEQ IDNO:63) 2R, AE003795 in cluster mir-3 to mir-6 miR-6UAUCACAGUGGCUGUUCUUUUU (SEQ ID NO:64) 2R, AE003795 in cluster mir-3 tomir-6 with 3 variants miR-7 UGGAAGACUAGUGAUUUUGUUGU (SEQ ID NO:65) 2R,AE003791 homologs: human, chr. 19 AC006537, EST BF373391; mouse chr. 17AC026385, EST AA881786 miR-8 UAAUACUGUCAGGUAAGAUGUC (SEQ ID NO:66) 2R,AE003805 miR-9 UCUUUGGUUAUCUAGCUGUAUGA (SEQ ID NO:67) 3L, AE003516homologs: mouse, chr. 19, AF155142; human, chr. 5, AC026701, chr. 15,AC005316 miR-10 ACCCUGUAGAUCCGAAUUUGU (SEQ ID NO:68) AE001574 homologs:mouse, chr 11, AC011194; human, chr. 17, AF287967 miR-11CAUCACAGUCUGAGUUCUUGC (SEQ ID NO:69) 3R, AE003735 intronic locationmiR-12 UGAGUAUUACAUCAGGUACUGGU (SEQ ID NO:70)  X, AE003499 introniclocation miR-13a UAUCACAGCCAUUUUGACGAGU (SEQ ID NO:71) 3R, AE003708mir-13a clustered with mir-13b  X, AE003446 on chr.3R miR-13bUAUCACAGCCAUUUUGAUGAGU (SEQ ID NO:72) 3R, AE003708 mir-13a clusteredwith mir-13b on chr. 3R miR-14 UCAGUCUUUUUCUCUCUCCUA (SEQ ID NO:73) 2R,AE003833 no signal by Northern analysis

TABLE 6 Human miRNA sequences and genomic location. From 220 short RNAssequenced, 100 (45%) corresponded to miRNAs, 53 (24%) to alreadycharacterized functional RNAs (rRNA, snRNAs, tRNAs), and 67 (30%)sequences with no database entry. For legend, see Table 1. chr. or EST,miRNA sequence (5′ to 3′) acc. nb. remarks* let-7aUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO:75)  9, AC007924, sequences of chr 9and 17 11, AP001359, identical and clustered with let-7f, 17, AC087784,homologs: C. elegans, AF274345; 22, AL049853 C. briggsae, AF210771, D.melanogaster, AE003659 let-7b UGAGGUAGUAGGUUGUGUGGUU (SEQ ID NO:76) 22,AL049853†, homologs: mouse, EST AI481799; ESTs, AI382133, rat, EST,BE120662 AW028822 let-7c UGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO:77) 21,AP001667 Homologs: mouse, EST, AA575575 let-7d AGAGGUAGUAGGUUGCAUAGU(SEQ ID NO:78) 17, AC087784, identical precursor sequences  9, AC007924let-7e UGAGGUAGGAGGUUGUAUAGU (SEQ ID NO:79) 19, AC018755 let-7fUGAGGUAGUAGAUUGUAUAGUU (SEQ ID NO:80)  9, AC007924, sequences of chr 9and 17 17, AC087784, identical and clustered with let-7a  X, AL592046miR-15 UAGCAGCACAUAAUGGUUUGUG (SEQ ID NO:81) 13, AC069475 in clusterwith mir-16 homolog miR-16 UAGCAGCACGUAAAUAUUGGCG (SEQ ID NO:82) 13,AC069475 in cluster with mir-15 homolog miR-17 ACUGCAGUGAAGGCACUUGU (SEQID NO:83) 13, AL138714 in cluster with mir-17 to mir-20 miR-18UAAGGUGCAUCUAGUGCAGAUA (SEQ ID NO:84) 13, AL138714 in cluster withmir-17 to mir-20 miR-19a UGUGCAUCUAUGCAAAACUGA (SEQ ID NO:85) 13,AL138714 in cluster with mir-17 to mir-20 miR-19b UGUGCAAUCCAUGCAAAACUGA(SEQ ID NO:86) 13, AL138714, in cluster with mir-17 to mir-20  X,AC002407 miR-20 UAAAGUGCUUAUAGUGCAGGUA (SEQ ID NO:87) 13, AL138714 incluster with mir-17 to mir-20 miR-21 UAGCUUAUCAGACUGAUGUUA (SEQ IDNO:88) 17, AC004686, homologs: mouse, EST, EST, BF326048 AA209594 miR-22AAGCUGCCAGUUGAAGAACUGU (SEQ ID NO:89) ESTs, human ESTs highly similar;AW961681†, homologs: mouse, ESTs, e.g. AA456477, AA823029; rat, ESTs,e.g. AI752503, BF543690 BF030303, HS1242049 miR-23 AUCACAUUGCCAGGGAUUUCC(SEQ ID NO:90) 19, AC020916 homologs: mouse, EST, AW124037; rat, EST,BF402515 miR-24 UGGCUCAGUUCAGCAGGAACAG (SEQ ID NO:91)  9, AF043896,homologs: mouse, ESTs, 19, AC020916 AA111466, AI286629; pig, EST,BE030976 miR-25 CAUUGCACUUGUCUCGGUCUGA (SEQ ID NO:92)  7, AC073842,human chr 7 and EST identical; EST, BE077684 highly similar precursorsin mouse ESTs (e.g. AI595464); fish precursor different STS: G46757miR-26a UUCAAGUAAUCCAGGAUAGGCU (SEQ ID NO:93)  3, AP000497 miR-26bUUCAAGUAAUUCAGGAUAGGUU (SEQ ID NO:94)  2, AC021016 miR-27UUCACAGUGGCUAAGUUCCGCU (SEQ ID NO:95) 19, AC20916 U22C mutation in humangenomic sequence miR-28 AAGGAGCUCACAGUCUAUUGAG (SEQ ID N0:96)  3,AC063932 miR-29 CUAGCACCAUCUGAAAUCGGUU (SEQ ID NO:97)  7, AF017104miR-30 CUUUCAGUCGGAUGUUUGCAGC (SEQ ID NO:98)  6, AL035467 miR-31GGCAAGAUGCUGGCAUAGCUG (SEQ ID NO:99)  9, AL353732 miR-32UAUUGCACAUUACUAAGUUGC (SEQ ID NO:100)  9, AL354797 not detected byNorthern blotting miR-33 GUGCAUUGUAGUUGCAUUG (SEQ ID NO:101) 22, Z99716not detected by Northern blotting *If several ESTs were retrieved forone organism in the database, only those with different precursorsequences are listed. †precursor structure shown in FIG. 4.

1. An isolated nucleic acid molecule selected from the group consistingof (a) the nucleotide sequence consisting of SEQ ID NO: 485, (b) anucleotide sequence consisting of the complement of (a), and (c) anucleotide sequence consisting of 18–25 nucleotides which has a sequenceidentity of at least 90% to SEQ ID NO: 485 or the complement of SEQ IDNO:
 485. 2. The nucleic acid molecule according to claim 1, wherein saidmolecule is a miRNA.
 3. The nucleic acid molecule according to claim 1,wherein said molecule is single-stranded.
 4. The nucleic acid moleculeaccording to claim 1, wherein said molecule is at least partiallydouble-stranded.
 5. The nucleic acid molecule according to claim 1,wherein said molecule is selected from the group consisting of RNA, DNAand modified nucleotide molecules.
 6. The nucleic acid moleculeaccording to claim 5, wherein said molecule comprises at least onemodified nucleotide.
 7. A recombinant expression vector comprising thenucleic acid molecule according to claim
 5. 8. The nucleic acid moleculeaccording to claim 1, wherein said nucleotide sequence in part (c) hasan identity of at least 95% to a sequence of (a) or (b).
 9. Acomposition comprising at least one nucleic acid molecule according toclaim 1 in combination with a pharmaceutically acceptable carrier. 10.The composition according to claim 9, wherein said pharmaceuticallyacceptable carrier is suitable for diagnostic applications.
 11. Thecomposition according to claim 9, wherein said pharmaceuticallyacceptable carrier is suitable for therapeutic applications.
 12. Thenucleic acid molecule according to claim 1, wherein said nucleotidesequence has a sequence identity of at least 90% to SEQ ID NO: 485 orthe complement of SEQ ID NO: 485, and further comprises a uridine (U) oradenosine (A).
 13. The nucleic acid molecule according to claim 6,wherein said modified nucleotide is a 2′ modified nucleotide.